CN105283792A - Method and optical arrangement for manipulating and imaging a microscopic sample - Google Patents
Method and optical arrangement for manipulating and imaging a microscopic sample Download PDFInfo
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- CN105283792A CN105283792A CN201480029470.9A CN201480029470A CN105283792A CN 105283792 A CN105283792 A CN 105283792A CN 201480029470 A CN201480029470 A CN 201480029470A CN 105283792 A CN105283792 A CN 105283792A
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- 230000003287 optical effect Effects 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000003384 imaging method Methods 0.000 title claims description 11
- 238000005286 illumination Methods 0.000 claims abstract description 157
- 238000005516 engineering process Methods 0.000 claims abstract description 12
- 230000005284 excitation Effects 0.000 claims abstract description 12
- 230000013011 mating Effects 0.000 claims description 29
- 238000001514 detection method Methods 0.000 claims description 14
- 230000033912 thigmotaxis Effects 0.000 claims description 6
- 238000012857 repacking Methods 0.000 claims description 4
- 239000003607 modifier Substances 0.000 claims 1
- 238000005452 bending Methods 0.000 description 11
- 238000006073 displacement reaction Methods 0.000 description 10
- 238000010586 diagram Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000386 microscopy Methods 0.000 description 3
- 230000002186 photoactivation Effects 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 238000002679 ablation Methods 0.000 description 2
- 201000009310 astigmatism Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000010304 firing Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000012576 optical tweezer Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/32—Micromanipulators structurally combined with microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
- G02B26/08—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light
- G02B26/0816—Optical devices or arrangements for the control of light using movable or deformable optical elements for controlling the direction of light by means of one or more reflecting elements
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- Optics & Photonics (AREA)
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Abstract
The invention relates to a method wherein a sample is manipulated by means of manipulation light and wherein the sample is imaged by means of SPIM technology with illumination with illumination light, in particular excitation light for the excitation of a fluorescence in the form of an illumination lightsheet. The method is distinguished by the fact that both the manipulation light and the illumination light are focused by the same objective, which is arranged in an objective operating position, or by different objectives that are brought successively into an objective operating position, and that the manipulation light and/or the illumination light, after passing through the objective, are/is deflected by means of a deflection device in such a way that said light propagates at an angle different from zero degrees relative to the optical axis of the objective.
Description
Technical field
The present invention relates to a kind of method, wherein operate sample by operational light, and wherein by means of SPIM technology imaging sample under the illumination of illumination light (exciting light especially for fluorescence excitation) using the form in illumination mating plate.
In addition the present invention relates to a kind of optical arrangement, its for operate sample and for by means of SPIM technology use in illumination mating plate form illumination light (exciting light especially for fluorescence excitation) illumination under imaging sample.
Background technology
The SPIM technology (monoplane illumination microscopy) of carrying out layering illumination sample wherein allows sensing image data more quickly and such as less than the spot type scanned samples impact of sample.A known art of the application of SPIM technology is the field of fluorescence microscopy, the fluorophore laser excitation in sample.In SPIM technology, excite and only occur over just in illumination light sheet (also referred to as " striation ").Avoid the damage of the sample produced by the illumination light in other plane thus.
The optical devices according to the operation of SPIM method are described in DE10257423A1.Use this microscopy, with thin striation illumination sample, the plane simultaneously perpendicular to illumination striation is observed.Illumination and detection are here via two independently beam path generations, and each beam path has independently optical device, particularly has two independent object lens perpendicular to one another.Striation is by illumination objective lens and by the cylindrical optical device generation be placed on before it.In order to image acquisition, sample moves by the striation static relative to detecting device, to use area detector layering to gather fluorescence and/or scattered light.Then thus obtained tomographic image data can be combined into the data set of the 3-D view corresponding to sample.The operation of the unexposed sample of the document.
DE20201110077U1 discloses a kind of layout for the sample that throws light under the background of SPIM microscopy.This layout comprises the light source for generating light beam, for generating the device of striation from light beam, and at least one object lens, at least one object lens described comprise and are embodied as and are intended to the detection light from sample directly or to be indirectly transported to the optical device of detecting device.In addition this layout comprise be positioned at object lens optical device after, for turning to the steering gear of striation.
SPIM technology and being combined in principle of optics sample operations are very attractive, and reason is the effect that the very fast SPIM technology with the impact of low sample is well suited for observing the operation of particularly larger, living organism.
From the known available additional operation light source of DE102007047464A1, its operational light is arranged via lens layout and catoptron and is coupled in illuminating beam path via illumination objective lens.Alternatively, same document additionally proposes operational light to be coupled in detection beam path by means of dichroic mirror, and it is directed on sample by objective lens.
Be transported in the embodiment of sample via illuminating beam path via detection beam path in the operational light of the type, three beam splitters and two diaphragms must insert in beam path arduously.The advantage of this setting is to operate from both direction.On the other hand, this causes complicated optical arrangement and at least causes light loss in illumination side.
Technical press " Selectiveplaneilluminationmicroscopytechniquesindevelopm entalbiology ", the people such as Huisken, Development136,1963-1975 (2009), disclose a kind of device of the type, wherein additionally provide its light to focus on light operate lasers on sample via objective lens.From " Three-dimensionallasermicrosurgeryinlight-sheetbasedmicr oscopy (SPIM) ", the people such as Engelbrecht, OpticaExpress6420, Vol.15, No.10 (2007), knows very similar layout.The article of the people such as Yanik, " TechnologiesforMicromanipulating, Imaging, andPhenotypingSmallInvertebratesandVertebrates ", Annu.Rev.Biomed.Eng.2011; 13:185-217, also disclose similar apparatus and method, wherein sample is pumped by kapillary.
Summary of the invention
So the object of the invention is to describe a kind of method, it allows general especially and sample operations and imaging samples flexibly.
This object is realized by a kind of method, it is characterized in that operational light and illumination light all by following focusing:
A. the identical object lens of object lens working position are arranged in, or
B. the different object lens of object lens working position are in succession brought to;
And after passing described object lens, operational light and/or illumination light turn to the angle making it become to be different from zero degree relative to the optical axis of described object lens to propagate by means of steering gear by this way.
Another object of the present invention is the optical arrangement describing a kind of aforementioned type, and it uses simple optical arrangement to allow general especially and sample operations and imaging samples flexibly.
This object is realized by a kind of optical arrangement, it is characterized in that operational light and illumination light all by following focusing:
A. the identical object lens of object lens working position are arranged in, or
B. the different object lens of object lens working position are in succession brought to;
And the steering gear of turning light light and/or operational light is in the downstream of described object lens being positioned at object lens working position.
The invention enables can very neatly and operate in the mode that can be adapted to application-specific individually and observe sample.Sample can be irradiated from different directions especially by operational light.Such as, also permission operational light from very not identical direction with one heart and (when needing) periodically interested same sample areas of reirradiation; This makes an explanation subsequently in detail.The present invention also make it possible to perform interested sample areas relevant, throw light on simultaneously.
Also particularly advantageously interested sample areas can be irradiated with the mode operational light being similar to the stereotaxis radiation used in such as the radiotherapy of oncotherapy.Can such as by by means of preferably adjustable suitable moving platform displacement sample positioning sample one's respective area in all three spatial directions, sample is arranged on described moving platform.Moving platform can be embodied as with maneuver mode adjustable especially.
The very special advantage that the present invention also has is that illumination light and operational light can irradiate sample from different directions.Therefore, such as, in the displacement of steering gear relative to object lens, and/or due to the use of the adjustment beam inflector assembly for operational light and/or illumination light, almost do not limit in the possible direction incided on sample and angle.Especially, user is not limited to only along the optical axis of object lens or irradiate sample perpendicular to the optical axis illumination light of object lens and/or operational light.
Also this point is considered especially, the very special advantage that the present invention has is that the effort can exempted to a great extent between long beamlet path switches and for being coupled into or a large amount of dichroic mirrors of decoupling operational light, even if in fact the versatility of arrangement according to the invention is greater than from the known layout of prior art.The instability certainly led to when that use multiple length, different beam path and fringe cost are avoided thus.
In fact in this respect advantageously at time repacking flying-spot microscope, particularly confocal scan microscope subsequently, to produce according to optical arrangement of the present invention and/or to perform according to method of the present invention, and large technical fee can be there is no.
Such as can perform micromanipulation that is very accurate, Wicresoft by means of optics sample operations.The character of the operational light such as in wavelength and/or optical power levels and/or intensity and/or in the geometric configuration of one or more operation light beam is preferably suitable for this application-specific.Such as, operational light can be pulse.Also non-pulse operational light can be used.
Arrangement according to the invention preferably comprises light source within this context, particularly LASER Light Source, and it is suitable for application-specific or is adapted to application-specific, particularly in aforesaid properties.Under the background of optical arrangement according to the present invention, such as, same lighting device may be used for generating run light and illumination light.But also may there is different light sources so that generating run light and illumination light.
Ultraviolet (UV) only such as may be used for cell ablation, DNA cuts and the high-energy light of microsection.But the common wavelength of 405nm also may be used for photoactivation and light conversion.Visible (VIS) light may be used for the bleaching experiment (fluorescence after FRAP-photobleaching recovers) of fluorescent dye.But visible ray also may be used for photoactivation and light conversion.Use infrared (IR) light, can by unexpected raised temperature (such as coolant-temperature gage), by introducing damage with Infrared irradiation with high degree of controlled, very low impact and local ways to restrain in very small size.Possible equally by organizing direct absorption.IR light also can be combined with optical tweezers.In addition, because biological structure is via multi-photon (MP) excitation principle and the interactional fact of infrared operational light, infrared operational light may be used for previously described process, as cell ablation, photobleaching, photoactivation and light conversion.
Advantageously, especially also can according to the present invention where necessary from obviously more than only both direction operation sample compared to from the known device of prior art.The use of adjustment beam inflector assembly (two the galvanometer mirrors be such as connected in series, deflects and a deflection in the Y direction in the X direction) allows such as to use spot, linear and free selectable region to operate illumination executable operations.Especially can in a controlled manner by irradiating the interested sample areas (ROI) of any shape of operation by operational light.
Steering gear such as can comprise one or more catoptron.Catoptron can be smooth especially.If if but such as illumination light and/or operational light move continuously along mirror surface and/or will additional focusing be obtained, then catoptron also can be bending.Alternatively or additionally, steering gear also may comprise at least one prism or at least one dichromatic beam splitter.
In a possible embodiment, predetermined operation light is focused on sample by the identical object lens being arranged in object lens working position with illumination light.Such embodiment makes to irradiate sample by operational light and illumination light especially simultaneously, and observes the sample reaction occurred fast after operation.
The special advantage that the embodiment of the type has is that the identical numerical aperture of object lens acts on illumination light and operational light; And when needing, therefore operational light can have disperse identical with illumination light.
But when identical object lens be used for operational light with for also in the time operational light be separated and illumination sample can be used during illumination light.
When irradiating discretely in time, operational light can be caused especially to be directly incident on sample after passing object lens, what do not have steering gear to produce turns to, but once illumination light is through object lens, it turns to the angle, the very especially propagation at a right angle that make it be greater than 10 degree relative to angle, especially one-tenth that the optical axis of object lens becomes to be different from zero degree by this way with steering gear.But here must consider, illumination light and the focus of operational light are not in from the identical spacing of object lens; This such as can by compensate lens displacement one amount, this amount equal when with operational light and with illumination between switch time along the difference of the spacing of optical axis.
When with operational light and with illumination between switch time also can switch between different object lens, introduce object lens working position, to avoid the problem of aforesaid spacing difference in particular by by corresponding required object lens.The refracting power of object lens is selected to make respective focal relative to object lens working position for the illumination light through steering gear with for being not identical through the illumination light of steering gear within this context by this way.
Do not consider this embodiment, because other reason also can realize the change of object lens, comprise and make by this way not to be by different refracting poweies but such as by displacement (above mention) the compensation spacing difference of object lens along optical axis.
Object lens can displaceability or mobility motor or realize by Mechanical Driven.Mobility such as on X, Y and/or Z-direction can realize by means of piezoelectric element or conventional Driving technique.
If such as want such as by means of spot, linear or free selectable region operation illumination, in the numerical aperture place executable operations higher than the numerical aperture for illumination light, according to a possibility embodiment of the present invention, this can realize like this: pivotable in the illumination objective lens with high numerical aperture to operate, and is pivoted back to the initial illumination object lens with lower numerical aperture to observe and to throw light on by illumination light.In order to allow to operate in normal illumination also occurent same focal point region, when must work as object lens change, perform the displacement along lighting optical axis.
The special advantage that the workability of high aperture objective has is that the conformation of illumination mating plate and/or illuminating bundle can be thin especially.Relative to SPIM imaging, increase resolution thus.
If such as want such as by means of spot, the illumination of linear or free selectable region, identical object lens (particularly having the object lens of high-NA) are used to perform illumination and operation, this can according to according to the present invention one may embodiment by by the object lens of focusing operation light and illumination light laterally (that is, transverse to optical axis) mobile scheduled volume, ensure that steering gear is still illuminated thus and realize.Its reason is that the object lens with high-NA are also typically the object lens that magnification is higher, namely has less scanning field.But if still want to arrange the big uncork of steering gear between steering gear and therefore larger object, illumination objective lens laterally must offset specified quantitative and make steering gear still be irradiated by scanning light beam.If the magnification of illumination objective lens increases by the factor of 2, then the transversal displacement of illumination objective lens must produce the following amount calculated: [(scanning field of lower magnification) deducts (scanning field compared with high power)] is divided by 2.If wanted not via steering gear but directly executable operations, and operate in focal plane while also via steering gear illumination, then object lens must move specified quantitative along optical axis; This amount is typically calculated as the half (in mm) of the opening of steering gear.If but hope operates other region of sample, also can use other value.
Cylindrical optical device such as can be used such as to generate mating plate from (such as, the laser) light beam with circular cross section.For the purposes of the present invention " cylindrical optical device " be understood to any astigmatism optical device and/or on a direction of the direction of propagation perpendicular to light than any optical device focused on more strongly on the other direction of the direction of propagation perpendicular to light.
But in very particularly advantageous embodiment, provide quasi-optical that illumination mating plate is made up of the illuminating bundle of movement continuously back and forth in mating plate plane.In order to these object optical devices such as can be included in the adjustable beam deflecting device in deflection angle aspect, use described beam deflecting device illuminating bundle preferably in illumination plane rapidly removable make illumination mating plate be in fact present in illumination light plane; And/or detecting from the detecting device of the light of sample with microscopical downstream evaluating apparatus with being provided for, described illumination undistinguishable is in such as with the continuous illumination mating plate that cylindrical optical device generates; And/or the data of generation can not or can not be distinguished with when throwing light on continuous illumination mating plate by view data substantially mutually that gather.
The beam deflecting device of the type such as can comprise at least one galvanometer mirror.Especially, such as, the beam deflecting device (described device under any circumstance exists) of flying-spot microscope (particularly confocal scan microscope) also can be used, if produce optical arrangement by the repacking of flying-spot microscope especially, if or performed according to method of the present invention by means of flying-spot microscope.
Especially when the shape of mating plate needs adjustable, particularly advantageously beam deflecting device comprises preferably at the multiple galvanometer mirrors especially for deflecting in orthogonal different deflection plane, such as X galvanometer mirror and Y galvanometer mirror, or other adjustable deflection device.Use the example utilizing steering gear to generate smooth quasi-optical especially, the advantage of multiple inflector assembly becomes obvious, described steering gear comprises one or more bending deviation mirror: iff passing through the bending deviation mirror of deflected illumination beam illumination in one plane, then then (track due to the illuminating bundle on bending deviation mirror necessarily bends) will cause unevenness but bending quasi-optical.But by being used in two the galvanometer mirrors deflected in different deflection plane, what the track of the illuminating bundle that can pull round by this way on mirror made to obtain is smooth quasi-optical.
Also quasi-optical that generates any configuration substantially can be controlled by the suitable of galvanometer mirror when (even if under the background using smooth deviation mirror) needs on the contrary.
The mating plate plane that turning light mating plate is propagated wherein preferably laterally aims at the optical axis of illumination objective lens and/or objective lens.
As discussed previously, advantageously can specify, be particularly used in the adjustable beam deflecting device in deflection angle aspect and illumination light and/or operational light are directed on steering gear via other optical element alternatively by object lens.
Especially, advantageously can specify, simultaneously or in a sequence use and have steering gear turning light light and/or the operational light of multiple steering device (particularly deviation mirror), described device is in the downstream of object lens being positioned at object lens working position.
Steering device can differently be arranged especially and/or differently aim at and/or belong to dissimilar.Such as can provide especially by means of beam deflecting device adjustable in deflection angle, operational light and/or illumination light are alternately directed on the steering device of different location and/or orientation, wherein operational light and/or illumination light redirect on sample by each steering device, particularly redirect on same sample region.Same sample areas can be irradiated from different directions thus by operational light.
This make especially can with one heart and/or stereotaxis be irradiated in same sample areas.Such as can irradiate interested sample areas consumingly by operational light thus, avoid peripheral region simultaneously, particularly because the impact of incident operational light is distributed in the outside of same area.The operational light that can such as make formation operate light beam thus especially moves on conical surface, be particularly used in the adjustable beam deflecting device in deflection angle aspect, and via the steering gear in the downstream at object lens, described steering gear comprises multiple steering device especially.
For application-specific, if such as will differently operate different sample areas, then can at operated device and/or can the power level of retouching operation light during beam deflection.
Steering gear also may comprise multiple steering device; Or be shaped by this way (particularly bending) makes illumination light and/or operational light to be directed to from different directions to be directed in interested sample areas on sample, particularly.
Detection light in a possible embodiment from sample passes the object lens of focus illumination light and/or is collimated by object lens.
But preferably, from sample detection light through be different from focus illumination light and/or operational light object lens objective lens and/or collimated by objective lens.
Especially, can advantageously specify, the optical axis of the object lens of focus illumination light and/or operational light and the optical axis of objective lens are aimed in parallel with each other and/or with collinear manner.The special advantage that the embodiment of the type has is optical devices can be configuration compact and sane especially, and specimen illumination region is especially easily close, therefore allows sample to introduce in specimen illumination region continuously fast and accurately.
Especially, can advantageously specify, illumination mating plate first in the vertical direction advances through illumination objective lens and then uses steering gear to redirect to horizontal direction so that a layer of the sample that throws light on.From illuminating layer light (particularly fluorescence) preferably in the vertical direction advance through objective lens.The configuration of the type makes it possible to the upright or inverted microscope platform of use standard to produce according to optical devices of the present invention.
In very particularly advantageous embodiment, the object lens of regulation focus illumination light and/or operational light and the steering gear that such as can comprise one or more deviation mirror are relative to each other arranged movably.Alternatively or additionally, on the object lens that steering gear also can be provided to be fastened on focus illumination light and/or operational light movably, and/or steering gear is fastened in objective lens movably.The advantage that these embodiments have easily regulates illumination light and/or operational light to be directly or via steering gear irradiate sample.In addition, by the adjustment of the relative position between steering gear and the object lens of focus illumination light and/or operational light, illumination light and/or operational light can be revised and incide direction on sample.
Alternatively or additionally, inflector assembly also rotatably can be arranged around objective lens and/or around the optical axis of objective lens, is particularly fastened in objective lens.Such embodiment makes such as easily can to regulate or revise illumination light and/or operational light and incides direction on sample.
Advantageously can specify, use illumination light and/or operational light to cause two-photon excitation.The pulsed laser for generating illumination light and/or operational light, particularly psec or femto-second laser can be advantageously comprised in this respect according to optical arrangement of the present invention.
As discussed previously, can be produced according to optical arrangement of the present invention by repacking flying-spot microscope (particularly confocal scan microscope).Also preferably can comprise according to optical arrangement of the present invention the generation that flying-spot microscope (particularly confocal scan microscope) makes independent of SPIM image by this way, described layout also can be used as flying-spot microscope.
As discussed previously, in special simple designs mode, steering gear can be arranged in objective lens, and described objective lens is arranged in the detection side of pick-up unit.This utilizes the existing parts of the form in objective lens dexterously to locate the necessary steering gear of generation mating plate securely thereon.Especially in order to high flexibility with simply adapt to the different requirements under the background utilizing optical arrangement, steering gear can be arranged in objective lens, especially in the removable mode of non-destructive.In other words, steering gear can be arranged in for the microscopic applications using illumination mating plate in objective lens, and can remove for different desired illumination from objective lens.In addition, the steering gear of different size can alternately use, and can easily change in the case of damages.
Steering gear such as can comprise catoptron and arrange, it preferably has multiple reflecting element and/or has at least one prism and/or at least one dichromatic beam splitter.Equally in a simple manner decoupled, steering gear can be arranged in the front side of objective lens.
Catoptron is arranged and can be comprised two reflecting elements that are smooth and mutual relative positioning in a simple manner decoupled for from two-side lighting and/or operation sample.In order to reliably avoid the shade in sample areas especially, catoptron arranges that can comprise can along can the multiple smooth or bending reflecting element arranged of the circular arc of predetermined length.Independent reflecting element can realize with simple reflex mirror annex, makes to pre-determine reflecting element relative positioning relative to each other when manufacturing catoptron annex.
Equally advantageously, the radius-of-curvature of bending reflecting element can be selected by this way to make all light beams from reflecting element reflection or can the light beam of predetermined quantity join a focus from reflecting element.Therefore most high beam load is positioned at this focus place; It can be located at the center of sample.In order to the focusing of the type at single focus place, radius-of-curvature equals the radius of the circular arc extending to the reflecting element arranged along circular arc from the center of sample.In order to use mating plate to throw light on, radius-of-curvature should be greater than two such radiuses from the center of sample to circular arc.
Accompanying drawing explanation
Theme of the present invention is schematically described in the accompanying drawings and is described with reference to accompanying drawing below, and the element of identical function indicates with identical Reference numeral.In the accompanying drawings:
Fig. 1 is the schematic side elevation of the exemplary embodiment according to optical arrangement of the present invention;
Fig. 2 is the schematic side elevation of the exemplary embodiment according to optical arrangement of the present invention, has the illumination objective lens of location different from the illumination objective lens in Fig. 1;
Fig. 3 is the schematic side elevation of the exemplary embodiment according to another optical arrangement of the present invention, and wherein illumination objective lens laterally offsets;
Fig. 4 schematically describes possible steering gear;
Fig. 5 is the schematic diagram of another possibility form describing steering gear;
Fig. 6 is the schematic diagram describing possible steering gear, and described steering gear has the multiple bending reflecting element arranged along circular arc; And
Fig. 7 describes to have multiple schematic diagram with another possibility steering gear of reflecting element that bends.
Embodiment
Fig. 1 is the schematic side elevation of the exemplary embodiment according to optical arrangement of the present invention, described optical arrangement for operate sample 1 and for use SPIM technology with in illumination mating plate 5 form illumination light (exciting light especially for fluorescence excitation) illumination under imaging sample.Illumination light generated by light source (not in the figure describe) and advance to adjustment beam inflector assembly 8 by beam splitter 10 and then via scanning lens 11 and tube lens 12 through be arranged in object lens working position, the entrance pupil 15 of the object lens 9 of focus illumination light.After passing object lens 9, illumination light turns to the angle making it become to be different from zero degree relative to the optical axis of object lens 9 to propagate by means of the steering gear 4 comprising deviation mirror 7 by this way.
Beam deflecting device 8 can be embodied as especially and deflect incident light independently of each other on two different directions (particularly X-direction and Y-direction).Such as, beam deflecting device 8 can comprise two galvanometer mirrors, and its turning axle is arranged in orthogonal plane.Alternatively, beam deflecting device 8 also such as can comprise universal installation mirror.
Only very the illumination mating plate 5 of schematic representation is preferably quasi-optical in the drawings, and it is generated by means of beam deflecting device 8 moves around rapidly by the illumination light of the form in illuminating bundle of launching from light source (description).Uniform strength distribution can be obtained thus especially.But also can produce illumination mating plate 5 by means of astigmatism optical device.
The detection light carrying out the layer of the sample of personal illumination light sheet 5 illumination uses pick-up unit 3 to detect.Detect light to collimate by means of objective lens 6, and be then imaged onto on detecting device 14 by means of optical device 13, described detecting device such as can be embodied as area detector, particularly CCD camera or the sensor based on CMOS.Detector maturation electric signal, described electric signal may be used for (alternatively after electronic processing) and is presented on a monitor by sample.The two dimensional image that can be combined into three-dimensional depiction in a large number can be obtained continuously by moving sample along the optical axis of object lens.
In like fashion, such as, first can obtain sample or at least one sample layer or interested especially sample areas the first image, then to perform (as described below) operation, can by again visible to generate another image after the effect of this operation.
Optical devices comprise another light source 2 of firing operation light.The operational light generated by light source 2 redirect to beam deflecting device 8 by beam splitter 10 and then advances to the object lens 9 being arranged in object lens working position via scanning lens 11 and tube lens 12.Object lens 9 are focusing operation light also.But beam deflecting device 8 does not resemble previously on one that operational light is directed to illumination light in deviation mirror 7, but guide by this way and make through its direct irradiation sample 1 after object lens 9.
The following fact must be considered here: the focus of illumination light and operational light has the diverse location in sample, make previously the focus of operational light can not be used to operate with the region of the focal illumination of illumination mating plate.
This is by correcting, as shown in the double-head arrow in figure along optical axis displacement object lens 9.Displacement stroke is preferably equally large relative to the initial difference of the spacing of object lens with focus.
Fig. 2 display is along with sample is to the necessary position of increase executable operations of the spacing of object lens.But as described in detail above, also can realize by different way compensating, such as such as, by using additional optical components (in the part of the beam path especially distinguishingly uniquely propagated in operational light, between another light source 2 and beam splitter 10) wherein and/or being undertaken throwing light on and operating by the different object lens of use.
Once operate, object lens can back be shifted again again to generate the image of sample (particularly by operation sample areas) in above-mentioned mode.
Steering gear 4 is arranged in objective lens 6.Steering gear 4 comprises two deviation mirrors 7 to allow selectively to throw light on from both sides sample.This allows to throw light on structure from side especially, this structure example as can not from above opposite side be radiated at owing to being covered by a part for sample self, or above being only radiated at deficiently.
Also can collimate another detection light 16 from sample in addition by means of the object lens 9 being positioned at object lens working position, and it is transported to detecting device (not describing in the drawings).Beam splitter 10 under this background is specialized by this way and detection light 16 can be passed through.
Fig. 3 is the schematic side elevation of the exemplary embodiment according to another optical arrangement of the present invention, and it allows also to use the object lens 9 with high-NA.The object lens with more high-NA typically have the advantage of high resolution capability, but have less visual field (scanning field).When layout shown in the figure, therefore specify, laterally (describe instruction for two by double-head arrow and object lens 9) even if mobile object lens 9 make also to irradiate steering gear when use has the object lens 9 of high-NA.Except transverse shift, preferably additionally perform the displacement along the optical axis of object lens 9 to keep illumination and to detect geometric configuration.
Replace two light sources being used for generating run light and illumination light independently, can alternatively provide by same light source generating run light and illumination light.This can be such as light source 2.Depend on application-specific, light source 2 can be embodied as and generate UV and/or IR and/or visible ray.
Fig. 4 to 7 shows the different exemplary embodiments of the steering gear 4 with different embodiment and the different deviation mirror 7 of layout and the possible beam path of operational light and/or illumination light in the planimetric map observed along the optical axis of object lens 9.
Fig. 4 is the schematic diagram that display has a possibility steering gear 4 of two deviation mirrors 7 be arranged opposite to each other.Deviation mirror 7 deflection is embodied as the illumination light of illumination mating plate so that the visual field 17 be positioned at least partially wherein of throw light on sample or sample.Visual field 17 is preferably corresponding to the region be imaged onto on detecting device (particularly two-dimensional detector).Alternatively or additionally, certainly also by means of deviation mirror 7, operational light can be directed on sample.
Fig. 5 is the schematic diagram describing to have another possibility form being all arranged in the steering gear of multiple deviation mirrors 7 of relative positioning side along circular arc.Also be similar to the steering gear shown in Fig. 6 and 7, the steering gear 4 of the type be suitable for very especially from different directions with one heart and/or stereotaxis be irradiated in sample areas.
Fig. 6 is the schematic diagram of the possibility steering gear 4 describing to have the multiple bending deviation mirror 7 arranged along circular arc.The radius-of-curvature of deviation mirror 7 preferably equals more than the twice of the radius r of circular arc.
Fig. 7 is that display has toward each other and along can the schematic diagram of another favourable steering gear 4 of bending deviation mirror 7 arranged of the circular arc of predetermined length.The radius-of-curvature of deviation mirror 7 corresponds to the radius of circular arc, makes the focusing of the light reflected by reflecting element 7 be present in the center of the circle be defined by a radius.All folded light beams are joined in center, and maximum load can be appeared in selected sample areas by stereotaxis illumination.Such as by by means of sample mounted thereto, preferably adjustable moving platform displacement sample suitably in all three spatial directions, sample areas can be positioned at center.Moving platform can be configured to maneuver mode adjustable especially.
The basis of steering gear shown in Fig. 5 to 7 can ensure the versatility of optical arrangement.With the concrete layout of independent steering component with configure irrelevant, each steering gear can be embodied as pre-packaged annex, especially for the pre-packaged annex being fastened to object lens and/or objective lens.Then the annex with mirror elements layout only needs to change to change between different applications.
In order to avoid repeating, about the other advantageous embodiment of optical arrangement according to the present invention, reader is see the summarized section described and subsidiary claim.
Finally, should notice that above-mentioned exemplary embodiment is only for the discussion of the instruction in claim clearly, instead of it is restricted to exemplary embodiment.
Claims (27)
1. a method, wherein operates sample by operational light, and wherein by means of SPIM technology imaging sample under the illumination light by the form in illumination mating plate, the exciting light especially for fluorescence excitation throw light on,
Wherein operational light and illumination light are all by following focusing:
A. the identical object lens of object lens working position are arranged in, or
B. the different object lens of object lens working position are in succession brought to;
And wherein after passing described object lens, operational light and/or illumination light turn to the angle making it become to be different from zero degree relative to the optical axis of described object lens to propagate by means of steering gear by this way.
2. method according to claim 1, wherein operational light and illumination light are directed on sample from different directions.
3. method according to claim 1 and 2, wherein:
A. described illumination mating plate quasi-optical of being made up of the illuminating bundle moved around continuously, or
B. described illumination mating plate is by quasi-optical that is used in illuminating bundle that the adjustable beam deflecting device in deflection angle aspect moves around continuously and forms, or
C. described illumination mating plate uses cylindrical optical device to generate.
4., according to the method one of claims 1 to 3 Suo Shu, wherein to throw light on sample by operational light and illumination light simultaneously.
5. according to the method one of claims 1 to 3 Suo Shu, wherein:
A. first by operational light and then to throw light on continuously in time sample by illumination light; Or
B. first by illumination light and then to throw light on continuously in time sample by operational light; Or
C. alternately to throw light on sample by illumination light and operational light.
6. according to the method one of claim 1 to 5 Suo Shu, wherein once illumination light is through described object lens, it turns to the angle, the very especially propagation at a right angle that make it be greater than 10 degree relative to angle, especially one-tenth that the optical axis of described object lens becomes to be different from zero degree by this way with described steering gear, and operational light is irradiated sample and do not turn to after passing described object lens.
7., according to the method one of claim 1 to 6 Suo Shu, wherein illumination light and/or operational light are used in the adjustable beam deflecting device in deflection angle aspect and are directed on described steering gear.
8. according to the method one of claim 1 to 7 Suo Shu, wherein:
A. described steering gear comprises multiple steering device, particularly multiple deviation mirror; And/or
B. described steering gear comprises multiple different and/or different arrange and/or different steering device, particularly multiple deviation mirror aimed at.
9. according to the method one of claim 1 to 8 Suo Shu, wherein:
A. sample is irradiated from multiple different directions, same sample areas particularly to be operated by operational light; And/or
B. with operational light with one heart and/or stereotaxis irradiate sample areas to be operated; And/or
C. beam deflecting device is used especially and via at least one steering gear in the downstream at described object lens mobile operational light forming operation light beam on conical surface; And/or
D. the power level of retouching operation light during operation and/or during beam deflection.
10. according to the method one of claim 1 to 10 Suo Shu, wherein:
A. the detection light from sample passes the object lens of focus illumination light and/or collimates with the object lens of focus illumination light; Or
B. the detection light from sample collimates through objective lens and/or by objective lens, and described objective lens is different from the object lens of focus illumination light and/or operational light.
11. methods according to claim 10, wherein the optical axis of the described object lens of focus illumination light and/or operational light and the optical axis of described objective lens are aimed in parallel with each other and/or with collinear manner.
12. according to the method one of claim 5 to 11 Suo Shu, wherein:
A. the described object lens of described steering gear and focus illumination light and/or operational light are relative to each other arranged movably; And/or
B. described steering gear is fastened on the described object lens of focus illumination light and/or operational light movably; And/or
C. described steering gear is fastened in described objective lens movably; And/or
D. described steering gear is rotatably arranged around described objective lens and/or around the optical axis of described objective lens.
13. according to the method one of claim 1 to 12 Suo Shu, wherein uses operational light and/or illumination light to cause two-photon excitation.
14. 1 kinds of optical arrangement, its for operate sample and for by means of SPIM technology with in illumination mating plate form illumination light, especially for fluorescence excitation exciting light illumination under imaging sample,
Wherein operational light and illumination light are all by following focusing:
A. the identical object lens of object lens working position are arranged in, or
B. the different object lens of object lens working position are in succession brought to;
And wherein at least one steering gear of turning light light and/or operational light is in the downstream of described object lens being positioned at object lens working position.
15. optical arrangement according to claim 15, wherein illumination light and operational light irradiate sample from different directions.
16. optical arrangement according to claim 15 or 16, wherein:
A. described illumination mating plate quasi-optical of being made up of the illuminating bundle moved around continuously, or
B. described illumination mating plate is by quasi-optical that is used in illuminating bundle that the adjustable beam deflecting device in deflection angle aspect moves around continuously and forms, or
C. there is the cylindrical optical device generating described illumination mating plate.
17. according to claim 14 to the optical arrangement one of 16 described, wherein can to throw light on sample by operational light and illumination light simultaneously.
18. according to claim 14 to the optical arrangement one of 17 described, wherein:
A. first by operational light and then to throw light on continuously in time sample by illumination light; Or
B. first by illumination light and then to throw light on continuously in time sample by operational light; Or
C. alternately to throw light on sample by illumination light and operational light.
19. according to the optical arrangement one of Claims 1-4 Suo Shu, wherein once illumination light is through described object lens, it turns to the angle, the very especially propagation at a right angle that make it be greater than 10 degree relative to angle, especially one-tenth that the optical axis of described object lens becomes to be different from zero degree by this way with described steering gear, and operational light is irradiated sample and do not turn to after passing described object lens.
20. according to claim 14 to the optical arrangement one of 19 described, and wherein illumination light and/or operational light are used in the adjustable beam deflecting device in deflection angle aspect and are directed on described steering gear.
21. according to claim 14 to the optical arrangement one of 20 described, wherein said steering gear comprises multiple different and/or different arrange and/or different steering device aimed at, particularly deviation mirror, to turn to the illumination light focused at the described object lens being arranged in object lens working position and/or the operational light focused at the described object lens being arranged in object lens working position.
22. according to claim 14 to the optical arrangement one of 21 described, wherein:
A. sample is irradiated from multiple different directions, same sample areas particularly to be operated by operational light; And/or
B. with operational light with one heart and/or stereotaxis irradiate sample areas to be operated; And/or
C. beam deflecting device is used especially and via at least one steering gear in the downstream at described object lens mobile operational light forming operation light beam on conical surface; And/or
D. the power level of power level modifier retouching operation light during beam deflection.
23. according to claim 14 to the optical arrangement one of 22 described, wherein:
A. the detection light from sample passes the object lens of focus illumination light and/or collimates with the object lens of focus illumination light; Or
B. the detection light from sample collimates through objective lens and/or by objective lens, and described objective lens is different from the object lens of focus illumination light and/or operational light.
24. optical arrangement according to claim 23, wherein the optical axis of the described object lens of focus illumination light and/or operational light and the optical axis of described objective lens are aimed in parallel with each other and/or with collinear manner.
25. according to claim 14 to the optical arrangement one of 24 described, wherein:
A. the described object lens of described steering gear and focus illumination light and/or operational light are relative to each other arranged movably; And/or
B. described steering gear is fastened on the described object lens of focus illumination light and/or operational light movably; And/or
C. described steering gear is fastened in described objective lens movably; And/or
D. described steering gear is rotatably arranged around described objective lens and/or around the optical axis of described objective lens.
26. according to claim 14 to the optical arrangement one of 25 described, wherein:
A. operational light and/or illumination light is used to cause two-photon excitation; And/or
B. there is pulsed laser, particularly femtosecond or picosecond laser, to generate illumination light and/or operational light.
27. according to claim 14 to the optical arrangement one of 26 described, wherein produces optical devices according to optical arrangement of the present invention by repacking flying-spot microscope, particularly confocal scan microscope; And/or described optical arrangement comprises flying-spot microscope, particularly confocal scan microscope.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE102013204959 | 2013-03-20 | ||
| DE102013204959.5 | 2013-03-20 | ||
| DE102013213781.8 | 2013-07-15 | ||
| DE102013213781.8A DE102013213781A1 (en) | 2013-03-20 | 2013-07-15 | Method and optical arrangement for manipulating and imaging a microscopic sample |
| PCT/EP2014/055661 WO2014147207A1 (en) | 2013-03-20 | 2014-03-20 | Method and optical arrangement for manipulating and imaging a microscopic sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105283792A true CN105283792A (en) | 2016-01-27 |
| CN105283792B CN105283792B (en) | 2018-09-21 |
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| CN201480029470.9A Active CN105283792B (en) | 2013-03-20 | 2014-03-20 | Method and optical arrangement for operating and being imaged micro- sample |
Country Status (6)
| Country | Link |
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| US (1) | US10012826B2 (en) |
| EP (1) | EP2976669B1 (en) |
| JP (1) | JP6580029B2 (en) |
| CN (1) | CN105283792B (en) |
| DE (1) | DE102013213781A1 (en) |
| WO (1) | WO2014147207A1 (en) |
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Also Published As
| Publication number | Publication date |
|---|---|
| JP6580029B2 (en) | 2019-09-25 |
| EP2976669B1 (en) | 2023-08-16 |
| US10012826B2 (en) | 2018-07-03 |
| CN105283792B (en) | 2018-09-21 |
| US20160048012A1 (en) | 2016-02-18 |
| EP2976669A1 (en) | 2016-01-27 |
| DE102013213781A1 (en) | 2014-09-25 |
| JP2016519331A (en) | 2016-06-30 |
| WO2014147207A1 (en) | 2014-09-25 |
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